Microencapsulation of oocytes for low-CPA (cryoprotectant) vitrification

说明（申请人提供）：卵母细胞冷冻保存对辅助生殖医学的发展、动物资源的维护和家畜管理具有重要意义。 然而，目前常用的卵母细胞冷冻保存方法无论是缓慢冷冻还是常规玻璃化冷冻都有固有的缺点。例如，缓慢冷冻（冷冻：液态水转变为冰晶）方法与由于冰形成和缓慢冷冻诱导的细胞脱水而导致的不可避免的细胞损伤相关。常规玻璃化（玻璃化：液态水转变成无定形的玻璃态而不是冰晶）方法所需的异常高的CPA（冷冻保护剂）浓度（4 - 7 M）即使在仅几分钟的短暴露时间内也会导致活细胞中的显著代谢和渗透损伤。据推测，正是这些固有的缺陷导致了迄今为止卵母细胞冷冻保存的惨淡结果。 在该R 01提案中概述的拟议研究的目标是通过将细胞微囊化在藻酸盐微胶囊中以在低CPA（低且无毒量的冷冻保护剂，= 1.5 M）浓度下玻璃化来开发用于细胞冷冻保存的新策略。所提出的低CPA玻璃化方法结合了当今常用的缓慢冷冻和常规玻璃化技术的所有优点，同时避免了它们的所有缺点。 自然繁殖（远交）的Peromyscus的卵母细胞将被用作本项目的生物模型，以便从拟议的研究中获得的结果可以更好地转移到实现其他自然繁殖的哺乳动物（包括人类）的卵母细胞的低CPA玻璃化冷冻。此外，鉴于胚胎冷冻保存总体上是成功的，因此本项目将使用Peromyscus胚胎作为基准生物模型来测试新方法。 据信，所提出的研究和新的低CPA玻璃化冷冻方法将对用于辅助生殖医学的卵母细胞冷冻保存、动物资源的维护和牲畜管理领域产生重大影响。 公共卫生相关性：卵母细胞冷冻保存对辅助生殖医学的发展具有重要意义。我们建议开发一种新的技术，以实现卵母细胞冷冻保存的性能大大提高。这项研究将对由于暴露于环境/职业危害或积极的医学治疗（如摘除性手术、放疗和化疗）而可能失去性腺功能的女性未来生育能力的保护产生重大影响。

DESCRIPTION (provided by applicant): Oocyte cryopreservation is of great importance to the advancement of assisted reproductive medicine, maintenance of animal resources, and livestock management. However, the commonly used methods today for oocyte cryopreservation by either slow-freezing or conventional vitrification have inherent drawbacks. For example, the slow-freezing (freezing: the transition of liquid water into ice crystal) approach is associated with inevitable cell injury due to ice formation and slow- freezing induced cell dehydration. The unusually high CPA (cryoprotectant) concentration (4 - 7 M) required by the conventional vitrification (vitrification: the transition of liquid water into an amorphous, glassy state rather than ice crystal) method can result in significant metabolic and osmotic injury in living cells even in a short exposure time of only a few minutes. Presumably, it is these inherent drawbacks that are responsible for the dismal outcome of oocyte cryopreservation to date. The goal of the proposed research outlined in this R01 proposal is to develop new strategies for cell cryopreservation by microencapsulating the cells in alginate microcapsule to vitrify at a low-CPA (low and non- toxic amount of cryoprotectants, = 1.5 M) concentration. The proposed low-CPA vitrification approach combines all the advantages of the commonly used slow-freezing and conventional vitrification techniques today while avoiding all their shortcomings. Oocytes of the naturally bred (outbred) Peromyscus will be used as the biological model in this project so that the results obtained from the proposed studies can be more transferable to achieve low-CPA vitrification of oocytes of other naturally bred mammals including humans. In addition, Peromyscus embryos will be used as the benchmark biological model in this project to test the new approach in view of the fact that embryo cryopreservation has been successful in general. It is believed that the proposed research and the novel low-CPA vitrification approach will have a significant impact on the field of oocyte cryopreservation for assisted reproductive medicine, maintenance of animal resources, and livestock management. PUBLIC HEALTH RELEVANCE: Oocyte cryopreservation is of great importance to the advancement of assisted reproductive medicine. We propose to develop a novel technology to achieve much improved performance for oocyte cryopreservation. This research will have a significant impact on the preservation of future fertility of women who may lose gonadal function because of exposure to environmental/occupational hazards or aggressive medical treatments such as extirpative surgery, radiation, and chemotherapy.